This invention relates generally to immune responses and more particularly to vectors containing immunostimulatory CpG motifs and/or a reduced number of neutralizing motifs and methods of use for immunization purposes as well as vectors containing neutralizing motifs and/or a reduced number of immunostimulatory CpG motifs and methods of use for gene therapy protocols.
Bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on peripheral blood mononuclear cells (PBMC) in vitro (Messina et al., J. Immunol. 147: 1759-1764, 1991; Tokanuga et al., JNCI. 72: 955, 1994). These effects include proliferation of almost all ( greater than 95%) B cells and increased immunoglobulin (Ig) secretion (Krieg et al., Nature. 374: 546-549, 1995). In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete predominantly Th 1 cytokines, including high levels of IL-12 (Klinman, D., et al. Proc. Natl. Acad. Sci. USA. 93: 2879-2883 (1996); Halpern et al. 1996; Cowdery et al., J. Immunol. 156: 4570-4575 (1996). These cytokines stimulate natural killer (NK) cells to secrete xcex3-interferon (IFN-xcex3) and to have increased lytic activity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., J. Immunol. 148: 4072-4076 (1992); Ballas et al., J. Immunol. 157: 1840-1845 (1996)). These stimulatory effects have been found to be due to the presence of unmethylated CpG dinucleotides in a particular sequence context (CpG-S motifs) (Krineg et al., 1995, supra). Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain CpG-S motifs (Tokunaga et al., Jpn. J. Cancer Res. 79: 682-686 1988; Yi et al., J. Immunol. 156: 558-564, 1996; Davis et al., J. Immunol. 160: 870-876, 1998).
Unmethylated CpG dinucleotides are present at the expected frequency in bacterial DNA but are under-represented and methylated in vertebrate DNA (Bird, Trends in Genetics. 3: 342-347, 1987). Thus, vertebrate DNA essentially does not contain CpG stimulatory (CpG-S) motifs and it appears likely that the rapid immune activation in response to CpG-S DNA may have evolved as one component of the innate immune defense mechanisms that recognize structural patterns specific to microbial molecules.
Viruses have evolved a broad range of sophisticated strategies for avoiding host immune defenses. For example, nearly all DNA viruses and retroviruses appear to have escaped the defense mechanism of the mammalian immune system to respond to immunostimulatory CpG motifs. In most cases this has been accomplished through reducing their genomic content of CpG dinucleotides by 50-94% from that expected based on random base usage (Karlin et al., J. Virol. 68: 2889-2897, 1994). CpG suppression is absent from bacteriophage, indicating that it is not an inevitable result of having a small genome. Statistical analysis indicates that the CpG suppression in lentiviruses is an evolutionary adaptation to replication in a eukaryotic host (Shaper et al., Nucl. Acids Res. 18: 5793-5797, 1990).
Nearly all DNA viruses and retroviruses appear to have evolved to avoid this defense mechanism through reducing their genomic content of CpG dinucleotides by 50-94% from that expected based on random base usage. CpG suppression is absent from bacteriophage, indicating that it is not an inevitable result of having a small genome. Statistical analysis indicates that the CpG suppression in lentiviruses is an evolutionary adaptation to replication in a eukaryotic host. Adenoviruses, however, are an exception to this rule as they have the expected level of genomic CpG dinucleotides. Different groups of adenovirae can have quite different clinical characteristics. Serotype 2 and 5 adenoviruses (Subgenus C) are endemic causes of upper respiratory infections and are notable for their ability to establish persistent infections in lymphocytes. These adenoviral serotypes are frequently modified by deletion of early genes for use in gene therapy applications, where a major clinical problem has been the frequent inflammatory immune responses to the viral particles. Serotype 12 adenovirus (subgenus A) does not establish latency, but can be oncogenic.
Despite high levels of unmethylated CpG dinucleotides, serotype 2 adenoviral DNA surprisingly is nonstimulatory and can actually inhibit activation by bacterial DNA. The arrangement and flanking bases of the CpG dinucleotides are responsible for this difference. Even though type 2 adenoviral DNA contains six times the expected frequency of CpG dinucleotides, it has CpG-S motifs at only one quarter of the frequency predicted by chance. Instead, most CpG motifs are found in clusters of direct repeats or with a C on the 5xe2x80x2 side or a G on the 3xe2x80x2 side. It appears that such CpG motifs are immune-neutralizing (CpG-N) in that they block the Th1-type immune activation by CpG-S motifs in vitro. Likewise, when CpG-N ODN and CpG-S are administered with antigen, the antigen-specific immune response is blunted compared to that with CpG-S alone. When CpG-N ODN alone is administered in vivo with an antigen, Th2-like antigen-specific immune responses are induced.
B cell activation by CpG-S DNA is T cell independent and antigen non-specific. However, B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995, supra). This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG-S DNA promotes antigen specific immune responses. The strong direct effects (T cell independent) of CpG-S DNA on B cells, as well as the induction of cytokines which could have indirect effects on B-cells via T-help pathways, suggests utility of CpG-S DNA as a vaccine adjuvant. This could be applied either to classical antigen-based vaccines or to DNA vaccines. CpG-S ODN have potent Th-1 like adjuvant effects with protein antigens (Chu et al., J. Exp. Med. 186: 1623-1631 1997; Lipford et al., Eur. J. Immunol. 27: 2340-2344, 1997; Roman et al., Nature Med. 3: 849-854, 1997; Weiner et al., Proc. Natl. Acad. Sci. USA. 94: 10833, 1997; Davis et al., 1998, supra, Moldoveanu et al., A Novel Adjuvant for Systemic and Mucosal Immunization with Influenza Virus. Vaccine (in press) 1998).
The present invention is based on the discovery that removal of neutralizing motifs (e.g., CpG-N or poly G) from a vector used for immunization purposes, results in an antigen-specific immunostimulatory effect greater than with the starting vector. Further, when neutralizing motifs (e.g., CpG-N or poly P) are removed from the vector and stimulatory CpG-S motifs are inserted into the vector, the vector has even more enhanced immunostimulatory efficacy.
In a first embodiment, the invention provides a method for enhancing the immunostimulatory effect of an antigen encoded by nucleic acid contained in a nucleic acid construct including determining the CpG-N and CpG-S motifs present in the construct and removing neutralizing CpG (CpG-N) motifs and optionally inserting stimulatory CpG (CpG-S) motifs in the construct, thereby producing a nucleic acid construct having enhanced immunostimulatory efficacy. Preferably, the CpG-S motifs in the construct include a motif having the formula 5xe2x80x2X1CGX23xe2x80x2 wherein at least one nucleotide separates consecutive CpGs, X1 is adenine, guanine, or thymine and X2 is cytosine, thymine, or adenine.
In another embodiment, the invention provides a method for stimulating a protective or therapeutic immune response in a subject. The method includes administering to the subject an effective amount of a nucleic acid construct produced by determining the CpG-N and CpG-S motifs present in the construct and removing neutralizing CpG (CpG-N) motifs and optionally inserting stimulatory CpG (CpG-S) motifs in the construct, thereby producing a nucleic acid construct having enhanced immunostimulatory efficacy and stimulating a protective or therapeutic immune response in the subject. Preferably, the nucleic acid construct contains a promoter that functions in eukaryotic cells and a nucleic acid sequence that encodes an antigen to which the immune response is direct toward. Alternatively, an antigen can be admininstered simulataneously (e.g., admixture) with the nucleic acid construct.
In another embodiment, the invention provides a method for enhancing the expression of a therapeutic polypeptide in vivo wherein the polypeptide is encoded by a nucleic acid contained in a nucleic acid construct. The method includes determining the CpG-N and CpG-S motifs present in the construct, optionally removing stimulatory CpG (CpG-S) motifs and/or inserting neutralizing CpG (CpG-N) motifs, thereby producing a nucleic acid construct providing enhanced expression of the therapeutic polypeptide.
In yet another embodiment, the invention provides a method for enhancing the expression of a therapeutic polypeptide in vivo. The method includes administering to a subject a nucleic acid construct, wherein the construct is produced by determining the CpG-N and CpG-S motifs present in the construct and optionally removing stimulatory CpG (CpG-S) motifs and/or inserting neutralizing CpG (CpG-N) motifs, thereby enhancing expression of the therapeutic polypeptide in the subject.